Insufficient
brightness of fluorophores poses a major bottleneck
for the advancement of super-resolution microscopes. Despite being
widely used, many rhodamine dyes exhibit sub-optimal brightness due
to the formation of twisted intramolecular charge transfer (TICT)
upon photoexcitation. Herein, we have developed a new class of quaternary
piperazine-substituted rhodamines with outstanding quantum yields
(Φ = 0.93) and superior brightness (ε × Φ =
8.1 × 104 L·mol–1·cm–1), by utilizing the electronic inductive effect to
prevent TICT. We have also successfully deployed these rhodamines
in the super-resolution imaging of the microtubules of fixed cells
and of the cell membrane and lysosomes of live cells. Finally, we
demonstrated that this strategy was generalizable to other families
of fluorophores, resulting in substantially increased quantum yields.
Rhodamine derivatives
and analogues have been widely used for different
super-resolution imaging techniques, including photoactivated localization
microscopy (PALM). Among them, rhodamine spirolactams exhibit great
superiority for PALM imaging due to a desirable bright–dark
contrast during the photochromic switching process. Although considerable
attention has been paid to the chemical modifications on rhodamine
spirolactams, the on-time of photochromic switching, one of the key
characteristics for PALM imaging, has never been optimized in previous
developments. In this study, we proposed that simply installing a
carboxyl group close to the lactam site could impose an intramolecular
acidic environment, stabilize the photoactivated zwitterionic structure,
and thus effectively increase the on-time. On the basis of this idea,
we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably longer on-time than the other tested
analogues, as well as an enhancement of single-molecule brightness,
an improvement on signal-to-noise ratio and an enlargement of total
collected photons of a single molecule before photobleaching. Finally,
super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a good temporal resolution
of 10 s, as well as a satisfactory localization precision of ∼25
nm. Through self-labeling protein tags, Rh-Gly modified
with a HaloTag ligand enabled super-resolution imaging of histone
H2B proteins in live HeLa cells; through immunostaining antibodies
labeled with an isothiocyanate-substituted Rh-Gly, super-resolution
imaging of microtubules was achieved in fixed cells. Therefore, our
simple and effective strategy provides novel insight for developing
further enhanced rhodamine spirolactams recommendable for PALM imaging.
Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.
Caged-fluorophores are potentially suitable for photo-activated localization microscopy (PALM) for super-resolution imaging. N-Nitroso is a simple and robust photo-cage with biocompatible nitric oxide as the only byproduct upon photolysis. We herein reported a novel PALM probe (NOR535) for super-resolution imaging of lysosomes with high localization precision.
A hallmark of cancer cells is a reversed transmembrane pH gradient, which could be exploited for robust and convenient intraoperative histopathological analysis. However, pathologically relevant pH changes are not significant enough for sensitive detection by conventional Henderson-Hasselbalch-type pH probes, exhibiting an acid-base transition width of 2 pH units. This challenge could potentially be addressed by a pH probe with a reduced acid-base transition width (i.e., Hill-type probe), appropriate p K, and membrane permeability. Yet, a guideline to allow rational design of such small-molecule Hill-type pH probes is still lacking. We have devised a novel molecular mechanism, enabled sequential protonation with high positive homotropic cooperativity, and synthesized small-molecule pH probes (PHX1-3) with acid-base transition ranges of ca. 1 pH unit. Notably, PHX2 has a p K of 6.9, matching the extracellular pH of cancer cells. Also, PHX2 is readily permeable to cell membrane and allowed direct mapping of both intra- and extracellular pH, hence the transmembrane pH gradient. PHX2 was successfully used for rapid and high-contrast distinction of fresh unprocessed biopsies of cancer cells from normal cells and therefore has broad potentials for intraoperative analysis of cancer surgery.
The
evolution of super-resolution imaging techniques, especially
single-molecule localization microscopy, demands the engineering of
switchable fluorophores with labeling functionality. Yet, the switching
of these fluorophores depends on the exterior conditions of UV light
and enhancing buffers, which is bioincompatible for living-cell applications.
Herein, to surpass these limitations, a nitroso-caging strategy is
employed to cage rhodamines into leuco forms, which for the first
time, is discovered to uncage highly bright zwitterions by green light.
Further, clickable construction grants the specificity and versatility
for labeling various components in living cells. The simultaneous
photoactivation and excitation of these novel probes allow for single-laser
super-resolution imaging without any harmful additives. Super-resolution
imaging of microtubules in fixed cells or mitochondria and the distribution
of glycans and H2B proteins in living cells are achieved at a molecular
scale with robust integrity. We envision that our nitroso-caging probes
would set a platform for the development of future visible-activatable
probes.
HoeSR, a nucleus specific probe for dSTORM super-resolution imaging of nucleus DNA in live cells, was designed by conjugating a rhodamine fluorophore and a Hoechst tag.
Live-cell single-molecule localization microscopy has advanced with the development of self-blinking rhodamines. A pK cycling of <6 is recognized as the criterion for self-blinking, yet a few rhodamines matching the standard fail for superresolution reconstruction. To resolve this controversy, we constructed two classic rhodamines (pK cycling < 6) and four sulfonamide rhodamines with three exhibited exceptional larger pK cycling characteristics (6.91−7.34). A kinetic study uncovered slow equilibrium rates, and limited switch numbers resulted in the reconstruction failure of some rhodamines. From the kinetic disparity, a recruiting rate was first abstracted to reveal the natural switching frequency of spirocycling equilibrium. The new parameter independent from applying a laser satisfactorily explained the imaging failure, efficacious for determining the propensity of self-blinking from a kinetic perspective. Following the prediction from this parameter, the sulfonamide rhodamines enabled live-cell super-resolution imaging of various organelles through Halo-tag technology. It is determined that the recruiting rate would be a practical indicator of self-blinking and imaging performance.
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